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Super-pangenome analyses across 35 accessions of 23 Avena species highlight their complex evolutionary history and extensive genomic diversity

Nature Genetics volume 57, pages 2276–2288 (2025)Cite this article

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Abstract

Common oat, belonging to the genus Avena with 30 recognized species, is a nutritionally important cereal crop and high-quality forage worldwide. Here, we construct a genus-level super-pangenome of Avena comprising 35 high-quality genomes from 14 cultivated oat accessions and 21 wild species. The fully resolved phylogenomic analysis unveils the origin and evolutionary scenario of Avena species, and the super-pangenome analysis identifies 26.62% and 59.93% specific genes and haplotypes in wild species. We delineate the landscape of structural variations (SVs) and the transcriptome profile based 1,401 RNA-sequencing (RNA-seq) samples from diverse abiotic stress treatments in oat. We highlight the crucial role of SVs in modulating gene expression and shaping adaptation to diverse stresses. Further combining SV-based genome-wide association studies (GWASs), we characterize 13 candidate genes associated with drought resistance such as AsARF7, validated by transgenic oat lines. Our study provides unprecedented genomic resources to facilitate genomic, evolution and molecular breeding research in oat.

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Fig. 1: Phylogeny, population structure and diverse phenotypes of cultivated oats and wild species in Avena.
Fig. 2: Inferred phylogeny, origin and evolution of Avena species.
Fig. 3: Super-pangenome of Avena and rich specific genetic resources in wild species.
Fig. 4: Characteristics of SVs among the 17 hexaploid oats.
Fig. 5: The impact of SVs on gene expression alteration.
Fig. 6: Identification and validation of key SVs and genes related to oat drought tolerance based on the SV-based GWAS.

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Data availability

The raw data (CRA015654 and CRA015559) and genome assemblies (GWHEROY00000000-GWHGDJH00000000.1) in this study have been deposited at the Chinese National Genomics Data Center (https://bigd.big.ac.cn/) under the BioProject accession number PRJCA024628. Raw sequencing data (SRR31832837–SRR31832882) and genome assemblies (JBKZJG000000000–JBKZJZ000000000 and JBKZKA000000000–JBKZKH000000000) have been deposited at the NCBI under the BioProject PRJNA1200805. Transcriptome sequencing data under normal growth (SRR31798633–SRR31798686 and SRR31814753–SRR31815091) have been deposited at the NCBI under the BioProject PRJNA1201252. Transcriptome sequencing data under different stresses (SRR31832975–SRR31833946) have been deposited at the NCBI under the BioProject PRJNA1203203. Whole-genome resequencing data of 1,078 hexaploid oat accessions were obtained from the OatOmics database (http://www.oatomics.com). Source data are provided with this paper.

Code availability

The scripts used in this study are available via GitHub (https://github.com/HongyuZhang-HBU/oat-pangenome) and Zenodo111 (https://doi.org/10.5281/zenodo.15761906).

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Acknowledgements

This work was supported by the Natural Science Foundation of Hebei Province (grant nos. C2023201074 and C2021201048 to H.D.), the Young Elite Scientists Sponsorship Program by CAST (grant YESS20210080 to H.D.), the National Natural Science Foundation of China (32100500 to H.D.), open funds of the State Key Laboratory of Plant Environmental Resilience (grant no. SKLPERKF2406 to Z.G.), the Interdisciplinary Research Program of Natural Science of Hebei University (grant no. 513201422004 to H.D.) and Post-graduate’s Innovation Fund Project of Hebei University (grant no. HBU2025BS011 to H.Z.). We sincerely thank Y. Fan at Chengdu University, Y. Liu at the Dingxi Academy of Agricultural Sciences and B. Wu at the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences for providing the oat accession resources. We also thank K. Wang at the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences for providing the TaWOX5 plasmid.

Author information

Author notes

  1. These authors contributed equally: Hongyu Zhang, Ningkun Liu, Yaru Wang, Xinyuan Zheng, Wei Li.

Authors and Affiliations

  1. College of Life Sciences, Institute of Life Science and Green Development, Hebei University, Baoding, China

    Hongyu Zhang, Ningkun Liu, Yaru Wang, Xinyuan Zheng, Wei Li, Ze Liu, Jianan Liu, Yu Wang, Longsheng Xing, Tao Li, Yange Yun, Qinghong Zhou, Meijia Wang, Yujie Qin, Jinjiang Yan, Zhizhong Gong, Qiang He & Huilong Du

  2. Hebei Basic Science Center for Biotic Interaction, Hebei University, Baoding, China

    Ningkun Liu, Wei Li, Qiang He & Huilong Du

  3. State Key Laboratory of Plant Environmental Resilience, College of Biological Sciences, China Agricultural University, Beijing, China

    Zhizhong Gong

Authors
  1. Hongyu Zhang
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  2. Ningkun Liu
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  3. Yaru Wang
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  4. Xinyuan Zheng
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  5. Wei Li
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  6. Ze Liu
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  7. Jianan Liu
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  8. Yu Wang
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  9. Longsheng Xing
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  10. Tao Li
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  11. Yange Yun
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  12. Qinghong Zhou
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  13. Meijia Wang
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  14. Yujie Qin
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  15. Jinjiang Yan
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  16. Zhizhong Gong
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  17. Qiang He
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  18. Huilong Du
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Contributions

H.D. conceived and supervised the project; H.D., Q.H. and Z.G. designed the study. Yaru Wang, X.Z., Q.H., Yu Wang and H.Z. sequenced and processed the raw data. Q.H., H.Z., Yaru Wang, M.W. and X.Z. assembled and annotated the genome. H.Z., X.Z., Z.L., Q.Z. and L.X. performed phylogenetic and genome evolution analyses; Yaru Wang, Q.H. and H.Z. conducted the transcriptome analysis; H.Z., Yaru Wang, Q.H., X.Z. and J.L. conducted pangenome analyses. Q.H., Yaru Wang, T.L., W.L. and N.L. performed TE and SV analyses. W.L., J.L. and Q.H. performed the population genetics analysis. N.L., Q.H., Y.Y., T.L., Y.Q. and J.Y. conceived of and designed the experiments. H.D. composed the outline of the paper. Q.H., W.L. and H.Z. wrote the paper. H.D. revised the paper.

Corresponding authors

Correspondence to Zhizhong Gong, Qiang He or Huilong Du.

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Competing interests

The authors declare no competing interests.

Peer review

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Nature Genetics thanks Mark Chapman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Geographical distribution and phenotype diversity of cultivated and wild oats accessions.

a, Principal component analysis (PCA) of 1,079 globally collected hexaploid oat accessions. PC1 (32.14%) clearly separates wild oat accessions (G1) and cultivated oat accessions (G2-G6); PC2 (19.45%) clearly separates G5 group of hulless oats from other groups; b, Geographic distribution of the 17 diverse representative accessions among 1,079 hexaploid oat accessions. The world map was generated using the WorldClim (https://worldclim.org/) whose data are freely available for academic use (https://worldclim.org/about.html). The color of points corresponds to Fig. 1a. c-e, The highly diverse agronomic phenotypes of representative wild oat accessions, including plant architecture (c), spike architecture (d) and spikelet (e).

Extended Data Fig. 2 Circos plot of the reference genome C0191 and assembly assessment of genomes in our study.

a, Circos plot showing chromosome-level features of C0191 reference genome. Tracks represent (I) chromosome length, (II) GC content, (III) gene density, (IV)TE density, (V) LTR density, (VI) DNA TE density, (VII) other TE density and (VIII) syntenic blocks. b, BUSCO completeness assessment for genome annotation of 35 oat accessions.

Source data

Extended Data Fig. 3 Phylogenetic relationships and karyotypes evolution of Avena species.

a, Distribution of Ks for orthologue duplicates identified between two (sub)genomes to estimate divergence time. Ad (A. damascene), As (A. brevis), D (A. maroccana-D), B (A. vaviloviana-B). b, SNP-based phylogenetic tree constructed with A. longiglumis as the reference genome based on our resequencing data and published resequencing data. c, A specific FISH probe targeting the As subtypes can label A. hispanica, A. brevis and A. nuda, but not A. hirtula, as observed in cells at the metaphase of mitosis. Scale bars, 5 μm. d, The reconstructed ancestral karyotype of the Avena genus using rice as the outgroup. Gene families with expansion and contraction in each species and node are highlighted in blue and orange, respectively. The red pentagrams represent the position of the inferred ancestor of A, B, C and D lineages. The arrows represented that the chromosome 1 and 4 in C subgenomes of tetraploids exhibited large-scale rearrangements. The red dashed boxes represent a large segment specific to the D subgenome of A. insularis but absent in that of A. maroccana and A. murphyi, then was translocated to the end of chromosome 2D during the process of polyploidization and retained in the hexaploid oats. e, The density of SVs in C1071, Afat, Aste, Aocc with the C0191 genome as reference. The average sequence similarity between subgenomes of C0191 and C1071, Afat, Aste, Aocc are shown in the heatmap. f, Proportions of contrasting gene tree topologies for 100 randomly selected single-copy genes (1,000 times) with regard to three major conflicting relationships. The values in the upper right corner of each tree show the percentage of windows that recovered the topology.

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Extended Data Fig. 4 The divergent genome composition between A. agadiriana and AABB tetraploid species.

a, Unsupervised hierarchical clustering of differential 15-mers validates that A. vaviloviana, A. barbata, A. abyssinica are successfully phased into two subgenomes based on clearly distinct patterns, while the two subgenomes of A. agadiriana showed higher sequence identity, making the subgenomes phasing relatively challenging and easily confused. b, The coalescent-based tree reconstructed from 100 randomly selected single-copy genes (1,000 times). The values in the upper right corner of each tree show the percentage of windows that recovered the topology. As (A. hispanica, A. brevis, A. atlantica, A. nuda). c, Intergenomic synteny among A. agadiriana, A. abyssinica, Avaviloviana and A. barbata. Some large inversions and translocations between A. agadiriana and species with AABB genome constitution were shown in blue. d, The average sequence similarity between the subgenomes of A. agadiriana, and three AABB species, as well as other subgenomes. e, Distribution of synonymous divergence (Ks) for orthologue genes identified between each subgenome of A. agadiriana (Aaga-Ac’ and Aaga-As’) and other subgenomes.

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Extended Data Fig. 5 Refined model for the origins, polyploidizations and evolutionary trajectories of Avena species.

a, Phylogenetic trees of 24 oat species with L. perenne and T. aestivum as an outgroup based on 26 chloroplast (cp) genomes and 7,137 mitochondrial (mt) SNP data sets. b-f, Divergence and polyploidization time estimation based on the 2,456 single-copy orthologous groups using MCMCTree, and distribution of synonymous divergence (Ks) for orthologue duplicates identified between two subgenomes.

Extended Data Fig. 6 Genetic diversity in cultivated and wild oats.

a, Venn diagram showing the numbers of common and unique gene families identified in panA, panC and panD of cultivated oats and panA, panB, panC and panD of wild oats. b, The distribution of CDS length in core, dispensable and private genes in cultivated and wild pangenomes. Cul-core (n = 1,460,017), Cul-dispensable (n = 394,369), Cul-private (n = 8,665), Wild-core (n = 776,159), Wild-dispensable (n = 553,189), Wild-private (n = 35,786). c, Proportion of genes with Pfam domains in core, dispensable, and private genes in cultivated and wild oats. Blue histograms indicate the genes with Pfam domain annotation; gray histograms indicate the gene without Pfam domain annotation. d, Comparison of gene expression levels in core, dispensable, and private genes in cultivated and wild oats. e-h, Comparison of the TE coverage with a window of 5 Mb (e), complete BUSCOs (n = 17) (f), Ka/Ks (n = 10,000) (g), NBS-encoding gene number (n = 17) (h) among the three subgenomes of hexaploid oats. i, The number of specific gene families derived from each wild oats compared to cultivated oats. The pie chart represents the proportion of gene families unique to wild oats that are derived from different number of subgenomes. j, A candidate introgression region from A. maroccana. The sequence similarity and SNP dataset all supported that A. maroccana showed close relationship with cultivated oats in this region.

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Extended Data Fig. 7 Verification and analysis of structural variations.

a, HiFi reads were used to validate the borders of 6 deletions randomly selected in C0080 and C0298. b, Schematic diagrams showed the distribution of inversion between two oat genomes (x-axis: C0191). Red box represented the validated inversion events relative to C0191. c, Illustration of inversion identified between two oat genomes by Hi-C contact map. Chromatin interaction heatmap revealed inversion signals appearing after manual flipping. These maps supported the inversions in chromosome 7D, 3 A, 5D and 3 C. d, Ten insertions were randomly selected for validation by PCR amplification. The detailed information of the validation of 10 SVs were presented in Supplementary Table 17. SV8, SV9 and SV10 came from other gels. Uncropped images are shown in Supplementary Figure 20. Different bands show the presence or absence of the SVs amplified in two oat genomes. The asterisk indicates the target band. The experiments were repeated independently three times, with similar results. e, Length distribution of deletions and insertions among 16 oat accessions. f, The haplotype of chromosome 4 A, 1 C, 2 C and 1D among C0355, C0298 and A. sterilis genomes indicated the similar genotype of C0298 with A. sterilis. g, Density of SV number per 100 bp in gene bodies and 3 kb flanking regions among 16 oat accessions.

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Extended Data Fig. 8 The effects of SVs on the gene expression.

a, Phylogenetic tree of RNA-seq samples. b, Principal-component analysis (PCA) for the RNA-seq samples based on the FPKM of each sample. c, The relative expression percentage of each subgenome based on the 1:1:1 (1:1) gene triads (pairs) in 18 oat accessions. The relative expression of different subgenomes of each accession summed to 100%. Error bar represents the SD of all RNA-seq samples of each accession. d, Comparison of average gene expressions between SV genes and no-SV genes among all RNA-seq samples in 16 oat accessions. e, The number of SV genes with suppression and promotion expression under normal growing and four different stress treatments.

Source data

Extended Data Fig. 9 Gene expression analysis under different stress treatments.

a, Heatmap of the expression levels of the genes under normal and different stress treatments of both leaf and root. b, Functional analysis of differential expression genes from four different stress treatments. c, RT-qPCR detection of A105905C025039.1 expression level in normal and drought conditions was shown in the left. Comparison of the expression level (FPKM) of A105905C025039.1 between the two haplotypes was shown in the right. d-f, RT-qPCR detection of A105902D033372.1, A105904C0105377.1 and A105902A0179076.1 expression level in control and different stress treatments in two tissues were shown in the left (Two-tailed Student’s t test). Error bar represents the SD of three biological replicates. Comparison of the expression level (FPKM) of A105902D033372.1, A105904C0105377.1 and A105902A0179076.1 between the two haplotypes in two tissues under corresponding stress treatment were shown in the right (two-sided Wilcoxon rank-sum test).

Source data

Extended Data Fig. 10 Functional validation of AsARF7.

a, The relationship among drought phenotype, SVs and gene expression. b, The expression heatmap of AsARF7 under ABA, drought and PEG6000 treatment. PEG600_S represents the aerial part after PEG600 treatment. PEG600_R represents the root part after PEG600 treatment. c, The expression of AsARF7 in different tissues including root, stem, leaf, flag-leaf, flower, fruit pod and seed. d, Detection of AsARF7 gene expression level in knockdown mutant strains. e, Water loss rate statistics of AsARF7 knockdown strains with three biological replicates. f, Chlorophyll content detection statistics of AsARF7 knockdown strains under drought treatment. g, Nuclear run-on assay showed that the transcription of AsARF7 was decreased in PEG6000 treatment plants. h, Genetic transformation in A. sativa. The up part: schematic representation of the pUBI::AsARF7-GFP vector with the UBI promoter and NOS terminator; The down part: detection of GFP fluorescence signal in callus induced from mature embryos infected with the pUBI::AsARF7-GFP vector (leaf). Regeneration phenotypes of mature embryos infected with the pUBI::AsARF7-GFP vector. Scale bar, 1 cm (right). Error bar represents the SD of three (c,d,g) of five (f) biological replicates.

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Zhang, H., Liu, N., Wang, Y. et al. Super-pangenome analyses across 35 accessions of 23 Avena species highlight their complex evolutionary history and extensive genomic diversity. Nat Genet 57, 2276–2288 (2025). https://doi.org/10.1038/s41588-025-02294-z

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